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. Author manuscript; available in PMC: 2010 Jul 13.
Published in final edited form as: J Cell Sci. 2008 Jun 17;121(Pt 14):2293–2300. doi: 10.1242/jcs.024018

A novel small molecule inhibitor reveals a possible role of Kinesin-5 in anastral spindle pole assembly

Aaron C Groen 1,2,*, Daniel Needleman 1,2, Clifford Brangwynne 2, Cristain Gradinaru 2, Brandon Fowler 3, Ralph Mazitschek 3, T J Mitchison 1,2
PMCID: PMC2902979  NIHMSID: NIHMS207309  PMID: 18559893

Summary

The tetrameric plus-end directed motor, kinesin-5, is essential for bipolar spindle assembly. Small molecule inhibitors of kinesin-5 have been important tools for investigating its function, and some are currently under evaluation as anti-cancer drugs. Most inhibitors reported to date are “non-competitive inhibitors” and bind to a specific site on the motor head, trapping the motor in a state with ADP bound, and a weak but non-zero affinity for microtubules. Here we used a novel ATP competitive inhibitor, “FCPT”, developed at Merck (USA) which competes with the ATP substrate. We found that it induced tight binding of kinesin-5 onto microtubules in vitro. Using Xenopus egg extract spindles, we found FCPT not only blocks poleward microtubule sliding but also induced loss of microtubules selectively at the poles of bipolar spindles (and not asters or monoasters). We also found that the spindle pole proteins, TPX2 and γ-tubulin became redistributed to the spindle equator, suggesting proper kinesin-5 function is required for pole assembly.

Introduction

Bipolar mitotic spindles composed of microtubules, motors, and other factors, are required for chromosome segregation (McDonald et al., 1979; McIntosh and Euteneuer, 1984; Salmon and Begg, 1980). In most spindles, a plus end directed, tetrameric motor from the kinesin-5 family (called Eg5 in Xenopus) is essential for spindle bipolarity (Cole et al., 1994; Heck et al., 1993; Kapitein et al., 2005; Sawin et al., 1992; Walczak et al., 1998). Kinesin-5 is proposed to function by sliding anti-parallel microtubules apart (Sharp et al., 1999). In Xenopus extract spindles, it is also proposed to drive continuous, poleward microtubule sliding during the metaphase steady state (Kapitein et al., 2005; Miyamoto et al., 2004).

An important sub-question for understanding spindle assembly is how spindle poles assemble, particularly in anastral spindles which lack pre-existing microtubule nucleating centers. Previous work has shown kinesin-5 has a role in organizing anastral spindle poles, as poles assembled without kinesin-5 appear as asters with large holes (“holey asters”) (Gaglio et al., 1996; Sawin et al., 1992; Sawin and Mitchison, 1994). However, analysis of such disorganized structures is complicated, making further conclusions about how kinesin-5 focuses microtubules into a spindle pole difficult.

Here, we used a novel ATP competitive inhibitor of kinesin-5, Compound-3—which we will now refer to as 2-(1-(4-fluorophenyl)cyclopropyl)-4-(pyridin-4-yl)thiazole or FCPT (Rickert et al., 2008). We found that FCPT induces a tight-binding of kinesin-5 onto microtubules and induced loss of microtubules selectively at the poles of Xenopus extract spindles without altering microtubule dynamics. We also found that FCPT blocks poleward microtubule sliding and redistributed the spindle pole proteins TPX2 and γ-tubulin. Our data suggest kinesin-5 may have a role in pole assembly in bipolar spindles by either directly or indirectly maintaining microtubule assembly.

Results and Discussion

Numerous small molecule kinesin-5 inhibitors have been described, and to date, all act by inhibiting the release of the ADP product (Cochran and Gilbert, 2005; Maliga et al., 2002). Members of this class are called “non-competitive inhibitors” and induce the formation of monpolar spindles in dividing cells. In this study, we used a representative non-competitive inhibitor, S-trityl-L-cysteine (STLC, see Figure 1A) (DeBonis et al., 2004) and a novel kinesin-5 inhibitor–FCPT–developed at Merck which competes with the ATP substrate ((Luo et al., 2007; Rickert et al., 2008); Figure 1A).

Figure 1. FCPT Induced Tight-Binding of Eg5 to Microtubules.

Figure 1

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A) The structures of kinesin-5 inhibitors: 2-(1-(4-fluorophenyl)cyclopropyl)-4-(pyridin-4-yl)thiazole (FCPT), GSK246053, monastrol, and S-Trityl-L-cysteine (STLC). B) FCPT, like AMP-PNP, induced binding of recombinant kinesin-5 motor domain to microtubules. The best-fit line of the dose-response curve identified the EC50 of AMP-PNP as 116 µM (+/− 17µM) and FCPT as 65 µM (+/− 10µM)—both in the presence of 1 mM ATP. Note: the concentration of FCPT required to bind 100% of the kinesin-5 motor domain to microtubules (1–2 mM) was above the solubility concentration for FCPT (approximately 500–600µM). (Error bars = Standard Error of the Mean.) C) Coomassie stained gels of lysates isolated from supernatants (S) and pellets (P) of either a DMSO (control), FCPT, or AMP-PNP treated kinesin-5 motor domain/microtubule mix (in the presence of 1 mM ATP) showing kinesin-5 and tubulin. D) FCPT enhanced the binding of kinesin-5 to microtubules. The best-fit line identified the apparent Kd of AMP-PNP (grey line) as 6.3µM (+/− 1.96) and FCPT (black line) as 1.7µM (+/−0.437)—both in the presence of 1 mM ATP, 8µM kinesin-5 and either 10µM FCPT or 10µM AMP-PNP. Fraction of kinesin-5 co-sedimenting with microtubules is scaled to reflect maximum binding as 1.0. (Error bars = Standard Error of the Mean.) E) Immunoblots show FCPT (compared to DMSO for a control) enhanced the binding kinesin-5 (approximately 3-fold), but not either XCTK2 or MCAK onto pelleted taxol polymerized microtubules in clarified Xenopus egg extracts. (Tubulin is a loading control.) F) FCPT inhibited kinesin-5 motility on Xenopus egg extracts spindles. The kymographs (measured from the red dotted line) of image sequences taken over 3.5 minutes (with 1 image every 3 seconds) show that X-rhodamine labeled kinesin-5 is not motile when FCPT (right; 200 µM) was present (shown as straight lines). In the control (left; same time frame as FCPT condition), kinesin-5 is dynamic, as speckles appear and disappear over time. White circle reflects outline of spindle. (Scale bar = 5µm)

FCPT Promoted Kinesin-5 Binding to Microtubules

FCPT showed good to excellent specificity for kinesin-5 inhibition compared to a panel of 8 kinesins and 36 kinases, suggesting it was suitable for cell biological experiments probing kinesin-5 function (Rickert et al., 2008). Inhibition of the microtubule stimulated ATPase activity of kinesin-5 motor domain by FCPT was competitive with the ATP substrate (Rickert et al., 2008), unlike non-competitive inhibitors (Maliga et al., 2002) (Cochran et al., 2004). The Ki for inhibition of ATPase activity was 110 nM (Rickert et al., 2008).

To test the effect of FCPT on the interaction between kinesin-5 and microtubules, we expressed monomeric motor domain, and performed co-sedimentation assays with microtubules. AMP-PNP was used as a control known to promote tight microtubule binding. We found both FCPT (EC50 ~65 µM +/− 10µM) and AMP-PNP (EC50 ~116 µM +/− 17µM) promoted a dose-dependent increase in the amount of motor domain co-sedimenting with microtubules, while very little motor domain co-sedimented in the presence of 1 mM ATP and no drug (Figure 1B–1C).

To calculate the apparent Kd, we performed co-sedimentation assays with varying concentrations of microtubules (Figure 1D). While the non-competitive inhibitors reduced the affinity of kinesin-5 for microtubules in the absence of ATP (Kd –without inhibitor = 0.7µM compared to Kd–with inhibitor = 2.3µM), we found FCPT only enhanced binding in the presence of ATP (data not shown), suggesting an aspect of the ATPase cycle is important for the activity of FCPT (Cochran et al., 2005). Co-sedimentation of microtubules and kinesin-5 required either FCPT (apparent Kd = 1.7µM +/−0.437) or AMP-PNP (apparent Kd = 6.3µM +/− 1.96µM), while very little kinsin-5 co-sedimented without drug (or AMP-PNP). Unlike AMP-PNP, the activity of FCPT was reversible upon resuspension of the microtubule pellet without drug (data not shown). These experiments show that FCPT, like AMP-PNP, locks the motor into a conformation with an increased affinity for microtubules. We will refer to this as “tight binding.”

To test the efficacy and specificity of the tight binding effect in cytoplasm, we added taxol to clarified Xenopus egg extract with or without FCPT, pelleted the assembled microtubule asters, and immunoblotted the pellets for motors involved in spindle morphogenesis. FCPT enhanced recruitment of kinesin-5 (by approximately 3-fold), while recruitment of MCAK, XCTK2, and dynein/dynactin were not affected (Figure 1E; data not shown).

To test if FCPT promoted tight binding of kinesin-5 to microtubules in spindles, we added small amounts (approximately 1 µM) of full length kinesin-5 labeled on random lysines with rhodamine (prepared as in (Kapoor and Mitchison, 2001) to pre-assembled Xenopus extract spindles, and imaged the resulting kinesin-5 speckles by spinning disc confocal microscopy. Without drug, the motor became transiently immobilized, as evidenced by short lines at various angles in the kymograph (Figure 1F, control). Addition of FCPT (at 200µM) greatly increased the recruitment of X-rhodamine-labeled kinesin-5 to spindles as quickly as we could assay (less than 2 minutes), and also caused the speckles to become immobilized for many seconds, as evidenced by the bright, vertical lines in the kymograph (1F, FCPT). Thus, the drug also promoted tight binding in the context of assembled spindles. These data also suggest poleward microtubule sliding is blocked, a point we confirm below with tubulin imaging.

FCPT Altered Spindle Morphology while Maintaining Bipolarity

FCPT and the non-competitive inhibitors of kinesin-5 each have dramatic, but different effects on spindle morphology in meiotic Xenopus egg extracts. As previously studied, non-competitive kinesin-5 inhibitors—such as STLC—induce the formation of monoaster spindles in dividing cells (Brier et al., 2004; Mayer et al., 1999; Mitchison et al., 2005)). Similar to other non-competitive kinesin-5 inhibitors, we found STLC addition caused pre-formed Xenopus egg extract spindles to collapse into monoasters over the course of 20 minutes, changing the average pole-pole length from 35 µm to approximately zero (using labeled anti-NuMA IgG to mark the poles; Figure 2A–2B) (Kapoor et al., 2000). The EC50 for STLC in this experiment was ~2µM (concentration of STLC required to assemble 50% monoasters), making it ~20× more potent than monastrol (Brier et al., 2004; DeBonis et al., 2004).

Figure 2. FCPT Decreased Spindle Pole Microtubule Density.

Figure 2

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A) FCPT treated (200 µM) Xenopus egg extract spindles did not collapse, while STLC (non-competitive kinesin-5 inhibitor) treated spindles collapsed within 20 minutes. Fixed images of different spindles at time points up to 20 minutes showing tubulin (red), NuMA (green), and DNA (blue). The spindle pole marker, NuMA (green) remained localized to the spindle pole in the presence of FCPT (Scale bar = 10µm.) B) FCPT treated Xenopus egg extract spindles maintained a constant length of about 35 µm, while STLC treated spindles collapsed to approximately 0 µm over 20 minutes. (Error bars = Standard Error of the Mean. N=6 for each time point). C) The ratio of the fluorescence of the spindle poles to mid-spindle decreased from approximately 0.9 to approximately 0.6 over 40 minutes (Error bars = Standard Error of the Mean. N=6 for each time point). D) Spindles assembled in the presence of FCPT are elongated and asymmetric, compared to controls. Fixed images of spindles showing tubulin (red), NuMA (green), and DNA. NuMA was diffusely localized on FCPT treated spindles (Scale bar = 10µm.)

FCPT addition to assembled spindles caused tubulin fluorescence near the spindle pole to decrease, over ~5 minutes, until ~50% of the initial amount remained (Figure 2A; Figure 2C), suggesting a decrease in microtubule density near spindle poles. Tubulin fluorescence near the equator remained approximately constant and unlike STLC addition, the pole-pole distance was not changed (Figure 2B). Despite the decrease in microtubule density at the poles, some pole structure remained, as evidenced by largely unchanged levels of anti-NuMA (Figure 2A). The EC50 of FCPT (concentration required to reduce the ratio of pole to equator fluorescence to approximately 0.8 or 50% of the maximum reduction) for promoting morphological change was ~75µM, and we used the compound at 200 µM in most experiments. This EC50 was higher than that for inducing tight binding with pure proteins, which is typical for hydrophobic drugs in extract, perhaps because much of the drug partitions into lipids. These data from fixed samples were confirmed by time-lapse imaging of drug-treated spindles and the morphological effects observed using labeled tubulin alone were identical in the absence of the anti-NuMA pole marker (data not shown).

The addition of FCPT before spindle assembly induced different morphological changes. While STLC addition–whether added before or after spindle assembly–induced the formation of monoaster spindles, FCPT inhibited spindle assembly and produced structures with disorganized, elongated microtubules (Figure 2D). Thus, FCPT–consistent with perturbing an essential meiotic kinesin, such as kinesin-5–affects spindle morphology whether added before or after spindle assembly.

FCPT Altered Spindle Pole Composition of Bipolar Spindles

We found that FCPT lowered microtubule density at spindle poles of bipolar spindles (Figure 2). To test whether FCPT also affects the protein composition of spindle poles, we measured the effect of FCPT on the localization of the spindle pole markers TPX2 and γ-tubulin. Each is required for assembly of microtubules during spindle assembly in extract (Gruss et al., 2001; Zheng et al., 1995). To visualize TPX2 and γ-tubulin, we used TPX2-GFP fusion protein and a non-perturbing, labeled γ-tubulin antibody (Figure 3A–B). FCPT caused TPX2 and γ-tubulin to relocalize from the poles to the band of remaining microtubules at the equator. The redistributions occurred over the same time-scale as the microtubule loss at the spindle poles (data not shown; Figure 2). Additionally, the kinesins XCTK2 and MCAK also disappeared from the FCPT treated spindle poles (at the same rate as tubulin), but unlike TPX2 and γ-tubulin neither were significantly enhanced in the spindle equator (Figure 3C–3D). We also found that FCPT did not enhance the binding of either TPX2, γ-tubulin, XCTK2, or MCAK to microtubules, as shown by western blot analysis of taxol polymerized microtubule pellets isolated from extracts (Figure 1E; Supplemental Figure 1A). Thus, FCPT may perturb the protein composition of intact poles of bipolar spindles by either affecting microtubule dynamics (transport, polymerization, or depolymerization) or directly changing the recruitment of spindle pole factors.

Figure 3. FCPT Redistributed TPX2 and γ-tubulin from the Spindle Pole of Bipolar Spindles.

Figure 3

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A–D) Fixed images of spindles treated with (and without) FCPT for approximately 20 mintues—tubulin (red), TPX2 (A; green), γ-tubulin (B; green), MCAK (C; green), XCTK2 (D). (Scale bar = 10µm.) A–B) FCPT redistributed the localizations of TPX2 and γ-tubulin from the spindle pole to the equator of the mid-spindle. C–D) FCPT removed the localization of MCAK and XCTK2 from the spindle pole. E) Fixed images of FCPT treated DMSO asters after (FCPT post-assembly) and before (FCPT pre-assembly) aster assembly–tubulin (red) and TPX2 (green). FCPT addition after DMSO aster formation (FCPT post-assembly) had no affect on the aster pole and localization of TPX2, while FCPT treatment before aster formation (FCPT pre-assembly) perturbed aster pole assembly and the pole localization of TPX2. (Scale bar = 10µm.)

Aster Poles did not Undergo FCPT Dependent Morphological Changes

To better understand the role FCPT has on spindle poles, we added FCPT to microtubule asters. We induced the formation of microtubules in extracts with DMSO, which assembled into aster-like structures after 30 minutes (Figure 3E). As shown previously, such structures localized NuMA, TPX2, and γ-tubulin (Groen et al., 2004; Wittmann et al., 1998; Wittmann et al., 2000). Similar to bipolar spindles, we found that the addition of FCPT to preformed asters induced the recruitment and immobilization of kinesin-5 to the aster (data not shown). FCPT addition before aster assembly abolished the assembly of all organized structures and mislocalized the pole markers, TPX2 and NuMA (Figure 3E; Supplemental Figure 2A). However, surprisingly, unlike bipolar spindles, we found that the addition of FCPT after aster assembly did not affect the aster structure and pole (Figure 3E). We found that the localization of pole markers such as NuMA, TPX2 and γ-tubulin were also not drastically changed (Figure 3E; Supplemental Figure 2A; data not shown). Similar effects were observed for RanGTP induced asters or STLC induced monoasters (Supplemental Figure 2B; data not shown). Our data suggest FCPT may require bipolarity (anti-parallel microtubules) to influence spindle pole structure.

Effect of FCPT on Microtubule Dynamics

Microtubules in extract spindles undergo poleward sliding, driven at least in part by kinesin-5 (Miyamoto et al., 2004). Non-competitive kinesin-5 inhibitors inhibit poleward sliding, so we wanted to test whether FCPT had similar effects. FCPT caused a dose-dependent decrease of the poleward microtubule sliding rate, measured by tubulin speckle imaging (Sawin and Mitchison, 1991; Waterman-Storer et al., 1998), with an EC50 of ~30µM (Figure 4A–B). Similar to the recruitment of rhodamine-labeled kinesin-5 to spindles, the inhibition of poleward sliding occurred as fast as we could assay (less than 2 min), suggesting the inhibition of poleward sliding is most likely due to perturbation of kinesin-5.

Figure 4. FCPT Inhibited Poleward Microtubule Movement and Spindle Pole Polymerization.

Figure 4

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A) FCPT inhibition of poleward microtubule movement was titratable. Localizations of speckle level Alexa-647 tubulin on Xenopus egg extract spindles with 0, 40, and 200 µM FCPT. The kymographs (measured from red dotted line) of image sequences obtained over the course of approximately 3.5 minutes (with 1 image every 3 seconds) show that poleward movement is only inhibited upon 200 µM FCPT addition. Intermediate concentrations of FCPT had intermediate microtubule poleward rates (see Figure 4B). Untreated control spindles had a microtubule poleward movement of approximately 1.9 +/− 0.2µm/min (+/− Standard Error of the Mean). White circles reflects outline of spindles. (Scale bar = 5µm.) B) The reduction of spindle pole density (as measured by the ratio of spindle pole to mid-spindle fluorescence) only occurred at FCPT concentrations (200 µM) sufficient to completely inhibit poleward microtubule movement. (Error bars = Standard Error of the Mean; N=3 for each point.) C) Alexa-488-EB1 localized on control and FCPT treated spindles. FCPT removed spindle pole localization of EB1 compared to controls (imaged approximately 4–5 minutes after FCPT addition; White circle reflects outline of spindle.). See Supplemental Movies 1–2. (Scale bar = 10µm.)

To determine the role FCPT had on microtubule polymerization at the spindle pole, we imaged directly labeled Alexa-488 EB1—a marker of microtubule polymerization—on control and FCPT treated spindles (Tirnauer et al., 2002b; Tirnauer et al., 2004). On control spindles, as shown previously, EB1 localized throughout the spindle—tracking along growing microtubule tips—suggesting polymerization occurs throughout the spindle (Tirnauer et al., 2002a). In FCPT treated spindles, EB1 disappeared from the spindle pole over a similar time-scale as the microtubule loss at the spindle poles (approximately 5 minutes) and remained localized to the band of microtubules at the equator with no significant change in over-all tip tracking dynamics in this region (Figure 4C; Supplemental Movies 1–2; data not shown). Thus, FCPT locally inhibits microtubule polymerization at the spindle pole of a bipole, but does not directly affect microtubule polymerization elsewhere.

Specificity of FCPT Effects

We found that the EC50 for poleward sliding inhibition (30µM) was lower than that for the morphological changes at spindle poles (~75µM; Figure 2; Figure 4B), suggesting the possibility of 2 targets with different binding affinities for FCPT. Though we cannot rule out this possibility, there are also many other equally possible explanations. For example, microtubule assembly (and/or the transport of assembly factors) may depend on poleward microtubule sliding and therefore the loss of microtubule assembly at the spindle pole may only occur when sliding is inhibited above a particular threshold, leading to a higher EC50 for the morphological changes. Consistent with this model, we the FCPT dependent inhibition of poleward sliding always occur (temporally) before the morphological changes at the spindle pole. Further experiments should address these models.

To further evaluate the specificity of the effects of FCPT on Xenopus extract spindles, we tested a structurally dissimilar compound that may act by a similar mechanism. Biphenyl sulfamide 20 (referred to here as GSK246053 (Figure 1A)) was developed as a specific ATPase competitive kinesin-5 inhibitor by Cytokinetics Inc and Glaxo Smith Kline Inc (Parrish et al., 2007). Mutations in the non-competitive site of kinesin-5, that block binding of non-competitive inhibitors, had no effect on ATPase inhibition by GSK246053, suggesting it binds at a novel site (Parrish et al., 2007). GSK246053 had the same effect as FCPT in the co-sedimentation assay in Xenopus extract and all of our spindle assays (Supplemental Figure 1B–1D). This similarity of effects of two structurally unrelated compounds, both developed as specific kinesin-5 inhibitors, provides confidence that our observed gain-of-function phenotypes result from successfully targeting the active site of kinesin-5.

Implications for Spindle Assembly

In this study, we found that an ATP-competitive inhibitor of kinesin-5, FCPT, reversibly binds the motor onto microtubules and produced many gain-of function phenotypes on extract spindles. Interestingly, we found it lowered spindle pole microtubule density in bipolar spindles, but not DMSO-induced asters or STLC monoasters. As asters and monoasters contain fewer anti-parallel microtubules than bipolar spindles (Heald et al., 1997; Kapoor et al., 2000; Mitchison et al., 2004), such experiments suggest the observed FCPT phenotypes may either require anti-parallel microtubules or that the poles of monoasters/asters and bipolar spindles rely on different assembly mechanisms. Further experiments are required to test this hypothesis.

From our data, it is not clear whether the reduction of EB1 at spindle poles reflects spatial changes in microtubule turnover (i.e. increase in catastrophes at spindle poles) or spatial changes in microtubule nucleation (i.e. less microtubule nucleation at spindle poles). The FCPT dependent loss of microtubule density at spindles poles could directly result from the inhibition of the transport of microtubules to the pole. Additionally, it could result from the inhibition/mislocalization of polymerization factors (such as TPX2, γ-tubulin) or the activation/mislocalization of depolymerization factors (such as Op18 or MCAK). Though, we did not observe the FCPT dependent recruitment of MCAK to the spindle pole (Figure 3C), there could be other unidentified depolymerization factors recruited to the spindle pole after FCPT treatment. Further experiments need to address these questions.

TPX2, γ-tubulin, and NuMA were previously argued to accumulate at poles due to dynein/dynactin mediated transport ((Buendia et al., 1990; Merdes et al., 1996; Verde et al., 1991; Wittmann et al., 1998)), a mechanism that should be insensitive to rigoring of kinesin-5. Interestingly, the pole localizations of TPX2 and γ-tubulin were perturbed by FCPT treatment, while the localization of NuMA was unaffected, suggesting TPX2 and γ-tubulin may depend upon other mechanisms—such as kinesin-5 dependent poleward sliding—to concentrate on spindle poles. While perturbation of dynein/dynactin does not inhibit poleward sliding (Maddox et al., 2003), the dynein/dynactin complex has a role in organizing microtubules into poles as well as transporting materials on them (Merdes et al., 1996; Wittmann and Hyman, 1999). However, distinguishing transport with dynein/dynactin from transport by attachment to sliding microtubules will be difficult, and we suspect both mechanisms are important. Overall, FCPT is an interesting tool for mitosis research, and a testament to the rich pharmacology of motor proteins.

Materials and Methods

Synthesis of FCPT and GSK246053

Synthesis of FCPT was performed with minor modifications according to the procedure reported in (Parrish and Dhanak, 2005).

1-(4-Fluorophenyl)cyclopropanecarbonitrile 10.00 g (7.4 mmol) (4-fluoro-phenyl)-acetonitrile and 9.56 mL (11.1mmol) 1,2-Dibromoethane were dissolved in 60mL Toluene followed by addition of 2.0 g (7.4mmol) tetra-N-butylammonium chloride and 60mL aqueous 12 M of Sodium hydroxide. The reaction mixture was stirred at room temperature. After 24 hours 200mL ethyl acetate was added, the organic layer was separated and washed twice with water. The combined organic layers were dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified on silica (Hexanes/ethyl acetate 5:1) to yield 5.80g (49%) of the desired product as yellow oil.

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1H NMR (300 MHz, CDCl3) δ7.30 (m, 2H), 7.06 (t, 2H, J = 8.6), 1.73 (q, 2H, J = 5.1), 1.38 (q, 2H, J = 5.3)

1-(4-Fluorophenyl)cyclopropanecarbothioamide 5.20 g (32.3mmol) 1-(4-fluoro-phenyl)-cyclopropanecarbonitrile, 7.22 g (35.5mmol) magnesium chloride hexahydrate and 10.8 g (19.4mmol) sodium hydrogen sulfide were dissolved in 100mL N,N-Dimethylformamide (Manaka, 2005). The reaction mixture was heated to 50°C. After 3h the reaction mixture was poured into 600mL water whereby a white precipitate forms. The aqueous layer was extracted three times with ether and the combined organic layers were washed with water. The combined organic layers were dried over Sodium sulfate and the solvent was removed under reduced pressure to yield an off-white fluffy solid, that was triturated twice with hexanes and dried under reduced pressure to yield 4.80g of the desired product as white powder.

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1H NMR (300 MHz, CDCl3) δ 7.85 (s, 1H), 7.40 (m, 2H), 7.09 (t, 2H, J = 8.6), 6.57 (s, 1H), 2.06 (q, 2H, J = 3.7), 1.32 (q, 2H, J = 3.7)

2-(1-(4-Fluorophenyl)cyclopropyl)-4-(pyridin-4-yl)thiazole (FCPT) 2.0g (10.2mmol) 1-(4-fluoro-phenyl-cyclopropanecarbothioic acid amide and 2.88g (10.2mmol) 2-bromo-1-(pyridin-4-yl)ethanone hydrobromide were suspended in 100mL Ethanol and heated to reflux for 2h. Upon cooling the product precipitated. The precipitate was filtered off and washed with cold ethanol and dried under reduced pressure to yield 2.44g (63%) of the desired product as hydrobromide (yellow powder) without additional purification.

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1H NMR (300 MHz, CDCl3) δ 8.85 (d, 2H, J = 6.4), 8.43 (d, 2H, J = 6.4), 8.11 (s, 1H), 7.47 (dd, 2H, J = 5.5, 8.3), 7.09 (t, 2H, J = 8.6), 1.86 (dd, 2H, J = 3.7, 6.3), 1.51 (dd, 1H, J = 3.8, 6.4).

Synthesis of GSK246053 was performed with minor modifications according to the procedure reported in (Coleman et al., 2005).

3-Fluoro-4'-(trifluoromethyl)biphenyl-4-amine: A solution of 1.6g tetrakis triphenylphosphane palladium in 10mL DMF was added to a solution of 6.61g (34.8mmol) 4-(trifluoromethyl)phenyl boronic acid and 5.51g (29mmol) 4-bromo-2-fluoro-anilin in 40mL DMF and 50mL 2M aqueous potassium carbonate. The reaction mixture was thoroughly purged with argon and stirred at 100C for 16h. After cooling to room temperature the reaction mixture was pored in 400mL of half saturated aqueous sodium bicarbonate solution and extracted three times with ether. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified on silica (20% ethyl acetate in hexanes) to yield 5.5g (72%) of the desired product as a white powder.

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1H NMR (500 MHz, CDCl3) δ 7.65 (d, 2H, J = 8.4), 7.61 (d, 2H, J = 8.4), 7.27 (dd, 1H, J = 1.8, 12.2), 7.22 (dd, 1H, J = 1.8, 8.2), 6.86 (t, 1H, J = 8.7), 3.90 (s, 2H)

GSK246053: 149uL of water was added to a solution of 718uL (8.25mmol) chlorosulfonyl isocyanate in 17.5mL acetonitrile at 0C. The reaction was stirred for one minute at 0°C and 3 hours 25°C. After cooling to 0°C a solution of 1.91g (7.5mmol) 3-fluoro-4’-(trifluoromethyl)biphenyl-4-amine in 17.5mL acetonitrile and 1.25mL pyridine was added dropwise. The reaction mixture was then warmed to room temperature. After 15 hours the reaction mixture was diluted with brine and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified on silica gel (35% ethyl acetate in hexanes) to yield 660mg (26%) of the desired product as white solid.

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1H NMR (500 MHz, CDCl3/CD3OD) δ 7.70 (t, 2H, J = 7.2), 7.65 (t, 2H, J = 8.3), 7.39 (dd, 2H, J = 5.7, 17.3), 6.71 (s, 1H),

Xenopus Egg Extract Spindles

Cycled Xenopus egg extract spindles were assembled from meiotic Xenopus egg extracts (CSF) as described previously (Desai et al., 1999; Murray, 1991). Briefly, spindles were assembled from CSF extract containing demembranated sperm nuclei (500/µl), cycled from interphase (with addition of 0.4mM CaCl2) for 80 minutes back to meiosis (with addition CSF). After spindle assembly (1–2 hours after CSF addition), spindle reactions were supplemented with various amounts of either FCPT (diluted in DMSO to 200 mM), 200 µM STLC (diluted in DMSO to 100mM) or 200µM GSK246053. Tubulin was visualized with addition of purified bovine tubulin (20µg/ml) directly labeled with X-rhodamine (Invitrogen) or Alexa-647 (Invitrogen) as described previously ((Hyman et al., 1991), (Sawin and Mitchison, 1991)). Generally, experiments were repeated 3–6 times using 3 different extracts.

Real-Time Imaging

Real time images were obtained using a 100×/1.4 NA Plan Apochromat objective (Nikon) on a Nikon TE 300 inverted microscope fitted with a Yokogawa spinning disk confocal head (Perkin Elmer) using an Andor EM-CCD camera (512 × 512 pixel sensor) driven by Andor software (Andor).

Real time images and fixed images were obtained using either a 20×/0.75 NA Plan Apochromat or a 40×/0.95 NA Plan Apochromat objective (Nikon) on an upright Nikon Eclipse 90i using an Orca ER cooled CCD camera (Hamamatsu) driven by Metamorph software (Molecular Devices).

NuMA and γ-tubulin were imaged by addition of 10µg/ml of Alexa-488 (Invitrogen) directly-labeled antibodies (Groen et al., 2004) after spindle assembly and FCPT treatment.

Recombinant EB1 was labeled with NHS-ester-Alexa488 (Invitrogen) and imaged by addition of 50nM to extracts before spindle assembly.

TPX2 was imaged by addition of approximately 20 nM of purified human GFP-TPX2 to extracts before spindle assembly. Human TPX2-GFP was purified from HeLA S3 suspension cells. Briefly, 1 L of cells was transfected using 750µl Lipofectamine (Invitrogen) with 500µg of purified pcDNA3.1-GFP-hTPX2 (a kind gift from S. Garrett and T. Kapoor, Rockefeller University). Lysates containing GFP-TPX2 were created by sonicating frozen pellets (created by pelleting and washing suspension cells 3 times in PBS, 1000 × g, 5 min each spin, 4˚ C) in 10× the volume of pellet in XB with no sucrose added (10mM HEPES, pH 7.7, 100 mM KCl, 1 mM MgCl2, 100 µM CaCl2) supplemented with 1 mM DTT and protease inhibitors (Roche Applied Scientific). The lysates were clarified (50,000 × g for 30 minutes) and GFP-TPX2 was purified first with a 5 ml HiTRAP S column (GE Healthcare) eluted with XB (no sucrose added) supplemented with 250 mM KCl. The fraction containing GFP-TPX2 was applied to a Superose-12 10/300 gel-filtration column (GE Healthcare) equilibrated with XB + 10% sucrose, 1 mM DTT. GFP-TPX2 containing fractions were frozen in liquid nitrogen.

Microtubule Pelleting Assay

To determine the FCPT EC50 for binding of kinesin-5 to microtubules in vitro Taxol (Sigma) stabilized microtubules (10 µM) and 4 µM clarified (200,000 × g for 20 minutes) gel-filtered (Superose-6 10/300 gel) recombinant HIS-kinesin-5 motor domain (amino acids 1–370; a kind gift from S. Kim and E. Wojcik, Louisana State University; Wojcik et al., 2004) were incubated with varying concentrations of either AMP-PNP, FCPT, or DMSO (control) for 5 minutes at room temperature. For the apparent Kd, varying concentrations of taxol stabilized microtubules were incubated with 8µM kinesin-5 and 10µM of either FCPT or AMP-PNP. All samples were pelleted through a 20% glycerol cushion (150,000 × g for 10 minutes). All buffers contained saturating amounts of ATP (1 mM ATP) in 1× BRB-80 plus 10µM taxol and the appropriate concentration of FCPT. The supernatant and the pellet of each reaction were supplemented with sample buffer for Coomassie stained SDS-PAGE. The fraction of kinesin-5 co-sedimenting with microtubules was determined using Image Pro (NIH). The best-fit curves (for the apparent Kd and EC50 calculations with a 95% confidence) were determined using Igor Pro (Wavemetrics) fitting to the equations: (kinesin-5 fraction bound) = c*x/(x+ EC50) and (kinesin-5 fraction bound) = c*x/(x+Kd), where x is the concentration of drug for EC50 measurements or concentration of tubulin for apparent Kd measurements. A correction constant (c) was necessary to fit each curve.

Taxol (10 µM; Sigma) induced microtubules from clarified Xenopus meiotic egg extracts (crude CSF spun 200,000 × g, 2 hrs.) containing either 200 µM FCPT or DMSO (control) were pelleted (20,000 × g, 10 min). The pellet was washed 3 times with 500 µl CSF-XB supplemented with 1 mM ATP, 1 mM GTP, 1 mM DTT, taxol (10 µM) and either 200 µM FCPT or GSK246053. The final pellet was resuspended in 20µl of sample buffer for use in western blot analysis probing against Eg5, XCTK2, MCAK, TPX2, γ-tubulin, and tubulin.

Image Analysis

Kymographs were taken from real-time image sequences using lines of 3 µm-width over maximum intensity particles on Metamorph (Molecular Devices). Average flux rates were calculated from manual tracking 10–15 particles on kymographs for 3–6 spindles from 3 different extract experiments for each condition.

The images from EB1 movies were aligned with StackReg plugin (Thévenaz et al., 1998).

The ratio of spindle pole to mid-spindle fluorescence was obtained by taking the average of the integrated intensity at each pole divided by the integrated intensity of the mid-spindle (within 1–3 µm of the chromatin) over a fixed rectangle for each spindle using ImageJ (NIH).

Supplementary Material

Figure S1
Figure S2
01

Acknowledgements

This project began during the 2006 Physiology Course at the Marine Biological Laboratory (MBL) at Woods Hole. We thank Physiology Course students C. Kilburn, C. Hentrich, E. Toprak, M. Bettencourt-Dias, C. Brawley, and M. Zuccolo, and all members of the Physiology Course 2006. We also thank T. Maresca, J. Gatlin, C. Field, and the MBL Cell Division Group. We thank Andor for loaning the EM-CCD camera (512 × 512 pixel sensor) and K. Hendricks (Nikon), H. Luther (MBL), and M. Peterson (MBL) for general equipment. We thank Dr. Paul Chang (MIT) for advice in TPX2-GFP purifications. T.J.M. was funded by CA078048-09 (National Cancer Institute).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1
NIHMS207309-supplement-Figure_S1.pdf (84.2KB, pdf)
Figure S2
NIHMS207309-supplement-Figure_S2.pdf (175.6KB, pdf)
01
NIHMS207309-supplement-01.pdf (83.7KB, pdf)

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